OUR TEAM

LAB 2 WRITE-UP

Thermal Cycler Engineering

Our re-design is based upon the Open PCR system originally designed by Josh Perfetto and Tito Jankowski.

New System Design
The two parts below that will be removed are the the knob and the bolt attached to it. These parts are present on the lid currently as a tightening mechanism but they will be removed to improve the design. On the side of the picture are the two heating plates present in the lid. To compensate for removing the tightening mechanism, the plates will be lowered to the point it makes contact with the samples in the heating block at the correct point in which the lid snaps into place.

Below is the top part of the general body of the PCR machine. It will be altered so buttons can be placed next to the LED screen.

Key Features
The design changes are predominately focused on the lid and the top body part next to the LED screen. Regarding the lid, the tightening mechanism was deemed ineffective due to not knowing how much to tighten the knob. To compensate for removing the mechanism, the heating plates will be lowered onto a specified height that it will make contact with the samples when the lid snaps into its natural place. Since sample containers are universally similar, one specified height will relinquish the need of having to set the heating plates themselves.

Design changes on the top body part will include changing the layout so that it may fit input buttons that will be connected and programmed internally later. These buttons will allow an individual to set up cycling details and will remove the need of an external computer to run the device.

Instructions

For the lid design

1. Remove the knob on the lid
2. Once the knob is removed, the bolt will be able to be removed.
3. Lower the bottom heating plate to the desired height
4. Lower the higher heating plate so the difference in space between the two plates was similar as before

For the top body part

1. Attach the part as you would normally
2. Connect the input buttons to the circuit board.

Protocols

Materials

Supplied in the Kit

Amount

PCR Machine (assembled)

1

Power Cord

1

Flourimeter Box

1

Flourimeter

1

Hydrophobic Slides

5

Supplied by User

Amount

Camera Phone

1

DNA Samples & Target Mix

As many as desired

Bulb pipettes

As many as desired

SYBR Green

As much as desired

Computer w/ ImageJ Software

1

PCR Protocol

DNA Measurement Protocol

1. One pipette was used to transfer each sample into an Eppendorf tube that contained 400ml of buffer. The number of the sample used was labeled clearly and all of the sample was transferred into this Eppendorf tube.

2. Two drops of SYBR Green I was placed on the first two center drops.

3. Next, two drops of the diluted sample was added on top of the SYBR Green I solution drops.

4. The drop was then aligned with the light on the flourimeter.

5. A smartphone placed on a stand was used to take as many pictures as needed of the sample and that picture was sent to a computer for analysis.

Research and Development

Background on Disease Markers

HIV is a virus that causes A.I.D.S. (acquired immunodeficiency syndrome). HIV affects the immune system by attacking specific cells that help the immune system work. These include the T Cells that help the white blood cells fend off bacteria, disease, and viruses. A SNP related HIV is rs1024611 and it affects Chromosome 17. http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=1024611

Primer Design

The primer that can be used to detect HIV is ATA and its reverse TAT. A diseased allele will only give results because of the way the PCR works and how the primers will attach. If the sample has the disease then the primer will attach to the DNA and as the PCR goes through its cycles there will be a multitude of the same DNA strand with the SNP rs1024611. If the sample does not have the disease allele then there will be no interaction with the DNA and the primer, therefore there will be no change from the original and the end results.